This application is a 371 of PCT/FR97/02331, filed Dec. 17, 1977, and claims priority to French application 96/16224, Dec. 17, 1996.
The present invention relates to the production by plants of recombinant collagens, in particular Type 1 homocatenary collagen [xcex1I (I)3] and other polypeptide derivatives, and their uses.
Patent WO 9603051 is known to the prior art which concerns the production of collagen in the milk of transgenic animals.
Collagen is an extracellular fibrous animal protein, widely found in animal tissues (recently detected in some mushrooms also). Some organs contain high quantities thereof: skin (at the dermis), tendons, bones. It is in fact a polymer whose remarkable properties are due both to the triple coil characteristics of some domains of its molecule and to the regularity of its supramolecular assemblies. It is involved in the organisation of the extracellular matrix grouping together twenty or so different molecules, called xe2x80x9ctypesxe2x80x9d that are identified by Roman figures (currently from I to XIX). The characteristic triple coil domain is formed by the coiling of three peptides, or xcex1 chains, arranged in a left wound coil and derived from a single conformation, the xcex1 coil of collagen. This specific conformation results from the repetition of a triplet of amino acids, Gly-X-Y in which X is frequently represented by proline and Y by hydroxyproline. These amino acids give stability to this type of coil. In a collagen molecule, the three xcex1 chains (identified by an index in Arabic figures) arranged in a right wound supercoil may be identical (xcex11 (or alpha1)), of two types (xcex11 and a2) or all different (xcex11, xcex12, xcex13). Collagen molecules therefore comprise helical domains (or collagen domains) and non-helical domains. They associate to form homo or heterotype polymers. Thus the collagen fibrils which form the essential part of the dermis are mostly made up of type I collagen [xcex11 (I)2 xcex12 (I)] associated with collagens of type III [xcex11 (III)3), of type V [xcex11 (V)2 xcex12 (V)] and covered by collagens of type XII (xcex11 (XII)3) and/or XIV [xcex11 (XIV)3]. Variants may exist: for example a homocatenary collagen of type 1 [xcex11 (I)3] is found in embryo tissues. During the biosynthesis of collagen some prolyl and lysil residues are hydroxylated, an addition of galactose possibly supplemented with a glucose may be made on some hydroxyl residues and conventional N and O glycosylations may occur on the non-helical domains. The recognition of the three chains forming a molecule and the start of their assembly are under the control of the C-terminal end (C-propeptide). Type I collagen undergoes enzymatic cutting of its non-helical ends subsequent to the previously cleaved peptide signal, the N and C-terminal propeptides are excised during maturation of the collagen leaving short, non-helical terminal extensions (telopeptides). It is these cleaved molecules which group together in arranged polymers (collagen fibrils) and which during the course of time undergo cross-linking via the hydroxylysyl residues of a molecule and the telopeptides of an adjacent molecule. The mechanical and biological properties of collagen have long been put to use; when cross-linked in irreversible manner (tanning process) it gives leather; when denatured by heating it gives rise to gelatin and glues. But it was only in the last decade that collagen truly provided biomaterials for pharmaceutical use (haemostatic compresses, sponges, dressings in particular healing dressings), medical use (prostheses such as cardiac valves, tendons and ligaments, skin substitutes, filling agents), odontological use (gum implants) and cosmetic use (additive, microcontainer for perfumed substances).
Later improved knowledge of this protein and of purification methods have led to the preparation of bovine and human placenta collagens in pre-defined form: gel, sponge, powder, suture and microsphere for example. Engineering of the extracellular matrices is also applied to the production of organoids containing transfected cells for gene therapy applications for example. The collagen mainly used is type I (generally associated with type III) for reasons of abundant availability and low purification costs, and the main sources have been bovine (skins unfit for tanning) ovine (hide and intestine) and human (placenta). This last source was exclusively reserved for pharmaceutical or medical applications.
Although the very useful mechanical and biological properties of collagen are clearly recognised, the use of this protein is questioned owing to the possible risks of contamination by non-conventional infectious agents. While the risks raised by bacterial or viral contamination can be fully controlled, this is not the case for those associated with agents of prion type. These infectious agents which appear to have a protein nature take part in the development of degenerative animal encephalopathy (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeld-Jacob disease, Gerstmann-Straussler syndrome, kuru). The long time of onset for their possible expression means that formal controls are difficult to conduct. These risks have already virtually frozen all marketing of human collagen, and the regulations laid down for the animal collagens concerned complicate purification processes and increase their cost.
Faced with these difficulties and radical deterioration in the image of mammalian collagen, one solution is the production of recombinant collagen which could be easily purified in a system that is not likely to give rise to pathogenic risks for man and whose industrial cost is not prohibitive. The inventors have therefore discovered and developed a production of collagen in plant species. For example we have been able to produce a human collagen of type I. Its molecule comprises a long, unbroken triple coil and is sparingly immunogenic after purification. The inventors have for example caused expression of the xcex11 (I) chain in order to obtain xcex11 (I)3 homocatenary molecules similar to those which exist in some tissues, especially embryo tissues.
Animal cells are, in theory, more adapted to the expression of mammalian genes. Their use however raises problems of protein maturation. The enzymatic equipment which carries out post-translational maturation differs from one tissue, organ or species to another. For example, it has been reported that post-translational maturation of a plasma protein may differ according to whether it is obtained from human blood or produced by a recombinant cell such as Chinese hamster ovary cells or in the milk of a transgenic animal. Moreover, the low levels of expression obtained with mammalian cells involve large volumes of in vitro cultures at high cost. With the production of recombinant proteins in the milk of transgenic animals (mice, ewes and cows) it is possible to reduce production costs and to overcome problems of expression level. However problems remain in respect of ethics and viral and subviral contamination (prions).
For these reasons, the transgenesis of mammalian genes in a plant cell could offer a pathway for the production in great quantities of new recombinant proteins at reduced production cost and with no risk of viral or subviral contamination. In 1983, several laboratories discovered that it is possible to transfer a heterologous gene into the genome of a plant cell, and to regenerate transgenic plants from these genetically modified cells. All the plants cells then have the genetically modified character transmitted to their descent by sexual fertilisation.
Through this work several teams have focused their attention on the production of recombinant mammalian proteins in plant cells or in transgenic plants (Barta et al., 1986; Marx et al., 1982). One of the first truly significant results in this area was the production of antibodies in transgenic tobacco plants. To express a heterologous protein in the grain, where plants stock proteins, Vandekerckhove""s team fused the sequence coding for leu-enkephaline to the gene coding for the 2S albumin of Arabidopsis thaliana. With this construct, transgenic Arabidopsis plants were produced which express leu-enkephaline specifically in the grains at expression levels in the region of 0.1% of total proteins. In 1990, the human albumin serum gene was transferred to tobacco and potato cells. Irrespective of the origin of the peptide signals (human or plant), levels of human albumin serum in the region of 0.02% of total proteins were obtained in particular in potato plant leaves. Other recombinant mammalian proteins have also been produced in plants: the surface antigen of hepatitis B, interferons, an anti-Streptococcus mutans mouse antibody, a caries agent, fragments of anti-cancer cell scFV antibodies, an anti-herpes antibody, cholera toxin and the human epidermal growth factor (E.G.F.). All this research has led to showing that the production of recombinant mammalian proteins in plant cells is possible, and that the mechanisms of protein synthesis from DNA sequences are similar in animal cells and plant cells. Numerous differences exist nonetheless between plant and animal cells, in particular in respect of the maturation of polymannosodic glycanns into complex glycanns, or at the cleavage sites of the peptide signals which means that it is not possible to guarantee that active or sufficiently active mammalian proteins can be obtained through plant cell transformation.
The inventors have discovered that the use of plant cells, transformed by an appropriate recombinant nucleotide sequence, can lead to obtaining collagen, in particular recombinant type I homotrimeric collagen [xcex11 (I)]3.
Another purpose of the invention is to provide the tools to implement such process, in particular new recombinant nucleotide sequences, cells of genetically transformed plants, plants or parts of plants (in particular leaves, stalks, fruits, seeds or grains, roots) that are genetically transformed, and fragments of these genetically transformed plants or parts of plants.
A further purpose of the invention is to provide new collagens produced by plants, in particular type I homotrimeric collagen [xcex11 (I)]3.
A further purpose of the invention is to provide new protein compositions able to be used for the implementation or supply of pharmaceutical, medical, odontological, cosmetic, biochemical or industrial compositions.
The invention concerns:
the use of a recombinant nucleotide sequence containing firstly a cDNA coding for one or more chains of mammalian collagen, in particular the one whose cDNA is that of the xcex11 collagen chain, or the derived proteins (by derived protein is meant any protein having at least 70% homology to the reference protein, in particular at least 80%, for example between 85 and 100% homology), and secondly the elements enabling a plant cell to produce the chain or chains of collagen or the derived proteins encoded by said cDNA, in particular a promoter and a transcription terminator recognised by the transcriptional machinery of the plant cells, for the transformation of plant cells with a view to obtaining from these cells or plants obtained from the latter, the chain or chains of collagen or the derived proteins, possibly in triple coil form,
a recombinant nucleotide sequence characterised in that it contains firstly the sequence coding for one or more chains of mammalian collagen, in particular that of the xcex11 collagen chain, or the derived proteins, and secondly the elements enabling a plant cell to produce the chain or chains of collagen or the derived proteins encoded by said sequence, possibly in triple coil form, in particular a promoter and a transcription terminator recognised by the transcriptional machinery of the plant cells,
a vector, in particular a plasmid, containing a nucleotide sequence of the invention inserted at a site that is non-essential for its replication,
a host cell, in particular any bacterium such as Agrobacterium tumefaciens, transformed by a vector of the invention,
a method of obtaining one or more collagen chains or derived polypeptides, optionally in triple coil form, characterised in that it entails:
the transformation of plant cells, in particular using a host cell of the invention, itself transformed by a vector of the invention, such as to incorporate into the genome of these cells a recombinant sequence of the invention,
optionally obtaining transformied plants from the above-mentioned transformed cells,
collecting the recombinant chain or chains of collagen or derived polypeptides produced in said above-mentioned transformed plant cells, in particular by extraction, optionally followed by purification,
a plant, plant extract or plant part, in particular genetically transformed leaves and/or fruits and/or seeds and/or plant cells, characterised in that it contains one (or more) recombinant nucleotide sequencers) of the invention incorporated into their genome in stable manner, these plants being chosen in particular from colza, tobacco, maize, pea, tomato, carrot, wheat, barley, potato, soybean, sunflower, lettuce, rice, alfalfa and beetroot.
one or more collagen chains or derived proteins characterised in that it is obtained in accordance with the method of the invention,
a collagen (in particular a collagen of type 1, II, III, IV or V) or a derived protein, characterised in that it is obtained in accordance with the method of the invention,
a product, in particular gelatin, characterised in that it is obtained from the collagen chains, collagen or their derived proteins of the invention,
a plant, plant extract or plant part, in particular genetically transformed leaves and/or fruits and/or seeds and/or plant cells, characterised in that they contain collagen chains, collagen or the derived proteins according to the invention, these plants being chosen in particular from colza, tobacco, maize, pea, tomato, carrot, wheat, barley, potato, soybean, sunflower, lettuce, rice, alfalfa and beetroot.
the use of plants, plant extracts or plant parts of the invention, and/or proteins (collagen chains, collagen or derived proteins) of the invention to obtain pharmaceutical, medical, odontological, cosmetic or biotechnological compositions,
a Biomaterial and a pharmaceutical, medical, odontological, cosmetic or biotechnological composition, characterised in that it comprises plants, plant extracts, plant parts or collagen chains, collagen or the derived protein of the invention,
One pharmaceutical composition of the invention particularly comprises any composition of the invention forming (or entering into the production of) a composition enabling the prevention or treatment of any pathology connected with collagen malfunctioning, or a compress for wound healing, a haemostat dressing, bedsore dressing, haemostat powder; or a dressing for burns.
One medical composition of the invention comprises in particular any composition of the invention forming (or entering into the production of) a corneal coating device, a coagulant film for organ resection (liver in particular), a filling material for the prevention of adhesions and fistulae, a composition for the fabrication of vascular and cardiac prostheses(valves), a guide-duct for nerve regeneration, bone and intra-body filling material, a container-releaser of active substances (hormones, growth factors, antibiotics, anticancer drugs, anti-inflammatories for example), a compression device (for example to reduce urinary incontinence), a skin substitute, surgical suture thread, an injectable material for cosmetic surgery (filling of skin depressions or face remodelling for example).
One odontological composition of the invention comprises in particular any composition of the invention forming (or entering into the production of) a gum dressing or a filling material.
One cosmetic composition of the invention comprises in particular any composition of the invention forming (or entering into the produciton of) a preparation additive (creams, ointments, make-up, liniments).
One medical composition of the invention comprises in particular any composition of the invention forming (or entering into the production of) a system for: covering cell culture dishes and bottles.
The invention also relates to a product, in particular gelatin, characterised in that it is obtained from collagen chains, collagen or their derived proteins according to the invention.
Gelatin is normally prepared from collagen by heating animal tissues (in particular skin and bones) in water and then drying (Parkany M, 1984). This preparation destroys the secondary structure of collagen and therefore leads to major changes in the product""s solubility and mechanical properties. Gelatin is the major constituent of glue. In cold water, it swells and is insoluble. In warm water it dissolves to give a very viscous solution which gels after cooling when the gelatin content is more than 1%. To prepare surgical prostheses, solutions containing 10 to 35% gelatin are used.
Gelatin""s property of being soluble in warm water makes it useful for numerous food and industrial applications. It is often prepared in powder from or in thin sheets. Depending upon the desired mechanical properties, glycerol, sorbitol or cross-linking agents may for example be added during the preparation of gelatin slabs.
Gelatin is also used in the bio-medical sphere, for sponges for example.
Advantageously, the recombinant nucleotide sequences of the invention contain one (or more) sequence(s) coding for a peptide responsible for addressing recombinant polypeptides to a determined compartment of the plant cell, in particular into the endoplasmic reticulum or the vacuoles, or even outside the cell in the pectocellulose wall, or in the extracellular space also called the apoplasm.
Among the transcription terminators able to be used for the transformation of plant cells under the present invention, mention may be made of the polyA 35S terminator of the Cauliflower Mosaic Virus (CaMV), or the polyA Nos terminator which corresponds to the 3xe2x80x2 region non-coding for the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens, nopaline strain.
In this respect, the object of the invention is any recombinant nucleotide sequence such as described above containing, upstream from said cDNA or its derived sequence, the polyA 35S terminator of CaMV or the polyA NOS terminator of Agrobacterium tumefaciens. 
Among the transcription promoters able to be used for the transformation of plant cells under the present invention, the following may be cited:
the 35S (P35S) promoter, or advantageously the double constitutive 35S promoter (Pd35S) of CaMV, these promoters allowing expression of the recombinant polypeptides of the invention in the entire plant obtained from cells transformed according to the invention, and are described in the article by Kay et al., 1987,
the PCRU promoter of the radish cruciferin gene allowing the expression of the recombinant polypeptides of the invention solely in the seeds (or grains) of the plant obtained from the cells transformed according to the invention, and described in the article by Depigny-This et al., 1992?
the PGEA1 and PGEA6 promoters corresponding to the 5xe2x80x2 region non-coding or the genes of the reserve protein of grains, GEA1 and GEA6 respectively, of Arabidopsis thaliana (Gaubier et al., 1993) and allowing specific expression in the rains,
the PSP super-promoter chimeric promoter (Ni M. et al., 1995), formed by the fusion of triple repetition of a transcriptional activator element of the promoter for the octopine synthase gene of Agrobacterium tumefaciens, of a transcriptional activator element of the promoter for the mannopine synthase gene, and of the mannopine synthase promoter of Agrobacterium tumefaciens, 
the actin promoter of rice followed by the actin intron of rice (APR-AIR) contained in the pActI-F4 plasmid described by McElroy et al. (1991),
the HMGW (High Molecular Weight Glutenine) promoter of barley (Anderson O. D. et al., 1989),
the promoter of the xcex3zein gene of maize (Pxcex3zein) contained in the pxcex363 plasmid described by Reina et al., (1990), and allowing expression in maize seed albumen.
In this respect, the object of the invention is any recombinant nucleotide sequence such as described above, containing upstream from said cDNA or its derived sequence the double 35S constitutive promoter (Pd35S) of CaMV, or the PCRU promoter of the radish cruciferin gene, or the PGEA1 or pGEA6 promoters of Arabidopsis thaliana, or the PSP super-promoter of Agrobacterium tumefaciens, or the APR-AIR promoter of rice, the HMGW promoter of barley or the pyzein promoter of maize.
The sequences coding for an addressing peptide used for the present invention may be of plant, human or animal origin.
Among the sequences coding for an addressing peptide of plant origin, the following may be cited:
the nucleotide sequence of 69 nucleotides (given in the following examples) coding for the prepeptide (peptide signal) of 23 amino acids of sporamine A in sweet potato, this peptide signal allowing entry of the recombinant polypeptides of the invention into the secretion system of the plant cells transformed according to the invention (namely, chiefly in the endoplasmic reticulum).
the nucleotide sequence of 42 nucleotides (given in the following examples) coding for the vacuole addressing N-terminal propeptide of 14 amino acids of sweet potato sporamine A, allowing the accumulation of the recombinant polypeptides of the invention in the vacuoles of the plant cells transformed according to the invention,
the nucleotide sequence of 111 nucleotides (given in the following examples) coding for the prepropeptide of 37 amino acids of sporamine A made up of, from the N-terminal part towards the C-terminal part, the 23 amino acids of the above-mentioned peptide signal, followed by the 14 amino acids of the above-mentioned propeptide, this prepropeptide permitting entry of the recombinant polypeptides of the invention into the secretion system and their accumulation in the vacuoles of the plant cells transformed according to the invention,
the three above-mentioned sequences being described in the articles by Murakami et al., 1986 and Matsuoka et al., 1991,
the carboxyterminal propeptide of barley lectin described in particular in the articles by Schroeder et al., 1993 and Bednarek et al., 1991,
and the PRS (Pathogenesis Related Protein, Comelissen et al. 1986) permitting secretion.
Mention may also be made, among the sequences coding for an addressing peptide, of that coding for the KDEL (SEQ. ID. NO.: 1), SEKDEL (SEQ. ID. NO.: 2), and HDEL (SEQ. ID. NO.: 3) peptides and allowing addressing in the endoplasmic reticulum.
A further object of the invention is any recombinant nucleotide sequence such as described above, containing a sequence coding for all or part of a vacuole-addressing peptide, in particular that of sporamine A in sweet potato, this sequence coding for a vacuole-addressing peptide being situated, in said recombinant nucleotide sequence, between the sequence coding for a peptide signal and that coding for said cDNA or its derived sequence, such that the first N-terminal amino acid of the vacuole-addressing peptide is bound to the last C-terminal amino acid of the peptide signal, and that the last C-terminal amino acid of said addressing peptide is bound to the first N-terminal amino acid of the polypeptide encoded by said cDNA or its derived sequence, in the protein encoded by said recombinant nucleotide sequence.
Yet a further object of the invention is any recombinant nucleotide sequence such as described above, containing a sequence coding for all or part of a vacuole-addressing peptide, in particular that of barley lectin, this sequence coding for a vacuole-addressing peptide being situated, in said recombinant nucleotide sequence, downstream from the sequence coding for said cDNA or its derived sequence, such that the first N-terminal amino acid of the vacuole-addressing peptide is bound to the last C-terminal amino acid of the polypeptide encoded by said cDNA or its derived sequence, in the protein encoded by said recombinant nucleotide sequence.